Summary
Two isoenzymes of citrate synthase were found in the endosperm of germinating castor bean seeds. One isoenzyme is restricted to mitochondria and the other to glyoxysomes. The two citrate synthases can be separated by (NH4)2SO4 gradient solubilization, eluting at 58 and 43% (NH4)2SO4, respectively. They are easily distinguished by the sensitivity to 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of oxalacetate: the glyoxysomal enzyme is completely inactivated within 15 seconds, while the mitochondrial enzyme remains unaffected. The time course of inactivation is a first order reaction. Oxalacetate prevents inactivation in high concentrations. The differences in DTNB sensitivity of the two citrate synthases can, in turn, easily be used to distinguish between the two isoenzymes. Since DTNB is a chromogenic compound in the assay for citrate synthase, it interfers with the assay at low concentrations of oxalacetate during Km determinations. This can be avoided by other assays which do not include DTNB. The inactivation of the glyoxysomal citrate synthase of castor bean endosperm is similar to the known inactivation of prokaryotic citrate synthases.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- DTNB:
-
5,5′-dithiobis(2-nitrobenzoic acid)
References
Axelrod, B., Beevers, H., 1972: Differential response of mitochondrial and glyoxysomal citrate synthase to ATP. Biochim. biophys. Acta256, 175–178.
Bajracharya, D., Schopfer, P., 1979: Effect of light on the development of glyoxysomal functions in the cotyledons of mustard (Sinapis alba L.) seedlings. Planta145, 181–186.
Barbareschi, D., Longo, P. G., Servettaz, O., Zulian, T., Longo, C. P., 1974: Citrate synthase in mitochondria and glyoxysomes of maize scutellum. Plant Physiol.53, 802–807.
Beevers, H., 1979: Microbodies in higher plants. Ann. Rev. Plant Physiol.30, 159–193.
Cazzulo, J. J., 1973: On the regulatory properties of a halophilic citrate synthase. FEBS Lett.30, 339–342.
Cooper, T. G., Beevers, H., 1969: Mitochondria and glyoxysomes from castor bean endosperm: Enzyme constituents and catalytic capacity. J. biol. Chem.244, 3507–3513.
Danson, M. J., Weitzman, P. D. J., 1973: Functional groups in the activity and regulation ofEscherichia coli citrate synthase. Biochem. J.135, 513–524.
— — 1977: Thiol groups ofEscherichia coli citrate synthase and their influence on activity and regulation. Biochim. biophys. Acta485, 452–464.
Gottschalk, G., Dittbrenner, S., 1970: Properties of (R)-citrate synthase fromClostridium acidi-urici. Hoppe-Seyler's Z. physiol. Chem.351, 1183–1190.
Greenblatt, G. A., Sarkissian, I. V., 1973 a: Citrate synthase of plants: sensitivity to sulfhydryl reagents and molecular weight of the enzyme. Physiol. Plant.29, 361–364.
— — 1973 b: Purification and characterization of citrate synthase fromPhaseolus vulgaris mitochondria. Phytochemistry12, 1249–1254.
Huang, A. H. C., Bowman, P. D., Beevers, H., 1974: Immunological and biochemical studies on isoenzymes of malate dehydrogenase and citrate synthase in castor bean glyoxysomes. Plant Physiol.54, 364–368.
King, T. P., 1972: Separation of proteins by ammonium sulfate gradient solubization. Biochemistry11, 367–371.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J., 1951: Protein measurements with the Folin phenol reagent. J. biol. Chem.193, 265–275.
Lück, H., 1965: Catalase. In: Methods of enzymatic analysis (Bergmeyer, H. U., ed.), pp. 885–894. New York: Academic Press.
O'Brien, R. W., Stern, J. R., 1969: Reversal of the stereospecificity of citrate synthase ofClostridium kluyveri by p-chloromercuribenzoate. Biochem. biophys. Res. Commun.34, 271–376.
Schnarrenberger, C., Oeser, A., Tolbert, N. E., 1971: Development of microbodies in sunflower cotyledons and castor bean endosperm during germination. Plant Physiol.48, 466–474.
— — —, 1973: Two isoenzymes each of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in spinach leaves. Arch. Biochem. Biophys.154, 438–448.
Srere, R. A., Brazil, H., Gonen, L., 1963: The citrate condensing enzyme of pigeon breast muscle and moth flight muscle. Acta chem. Scand.17, 129–134.
Srere, P. A., Kosicki, G. W., 1961: The purification of citrate condensing enzyme. J. biol. Chem.236, 2557–2559.
—,Pavelka, S., Das, N., 1971: An unusual citrate synthase from mango fruit. Biochem. biophys. Res. Commun.44, 717–723.
Weitzman, P. D. J., 1966: Reduced nicotinamide-adenine dinucleotide as an allosteric effector of citrate-synthase activity inEscherichia coli. Biochem. J.101, 44 c-45 c.
—,Danson, M. J., 1975: Citrate synthase. Current Topics Cell. Regulation10, 161–204.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Schnarrenberger, C., Fitting, K.H., Tetour, M. et al. Inactivation of the glyoxysomal citrate synthase from castor bean endosperm by 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). Protoplasma 103, 299–307 (1980). https://doi.org/10.1007/BF01276275
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01276275